人主动脉平滑肌细胞体外分离及培养

Isolation and culture of human aortic smooth muscle cells

目的探讨人主动脉平滑肌细胞(HAoSMC)的体外分离及培养方法。方法人升主动脉组织取自在北部战区总医院接受外科主动脉置换手术的A型主动脉夹层患者。无菌条件下撕除主动脉内膜,获得中膜,将中膜剪成约2 mm长的组织块,用0.15%的Ⅱ型胶原酶消化组织块10 h,吹打后接种于6孔板中,显微镜下观察培养不同时间点的细胞形态特征,免疫荧光染色法检测平滑肌细胞标志物SMα-actin及SM 22α的表达。结果胶原酶消化法可成功将平滑肌细胞从主动脉中膜分离出来。分离出的细胞在培养第1天开始贴壁,第7天呈现典型长梭形,第14天基本融合。培养的细胞SMα-actin及SM 22α染色阳性。结论本研究采用酶消化法成功从主动脉夹层患者标本中分离并培养出HAoSMC,为进一步阐明主动脉疾病的发病机制奠定了基础。

Objective To investigate the method of isolation and culture of human aortic smooth muscle cell(HAoSMC)in vitro.Methods Human aortic tissue was obtained from patients with type A aortic dissection who underwent aortic replacement in General Hospital of Northern Theater Command.The intima of the aorta was torn off under aseptic conditions to obtain the middle membrane.The middle membrane was cut into tissue blocks about 2 mm long.The tissue blocks were digested with 0.15%typeⅡcollagenase for 10 h,and then inoculated in 6-well plates after blown.The expression of SMα-actin and SM 22αin smooth muscle cells was detected by immunofluorescence staining.Results Smooth muscle cells were successfully isolated from the tunica media of the aortic tissue by collagenase digestion.The isolated cells began to adhere to the wall on the first day of culture,showed typical long shuttle type on the seventh day,and basically fused on the 14 th day.The cultured cells were positive for SMα-actin and SM 22αstaining.Conclusion HAoSMC was successfully isolated and cultured from human aortic tissue.The successful cultivation of HAoSMC is helpful for elucidating the pathogenesis of aortic diseases from the perspective of smooth muscle cells.